Peptide inhibitors of fibronectin and related collagen-binding proteins

ABSTRACT

Peptides derived from the second type 1 repeat of human endothelial cell thrombospondin which bind to the gelatin-binding domain of fibronectin have been isolated and synthetically produced. The peptides can be used to bind to fibronectin or other related collagen-binding proteins to inhibit fibronectin-dependent cell adhesion to collagen matrices and to inhibit interactions with collagen of other proteins that share homologies with the gelatin-binding domain of fibronectin.

FIELD OF THE INVENTION

The present invention relates to peptides derived from a domain ofthrombospondin mediating interaction of thrombospondin with fibronectin,which peptides bind specifically to fibronectin and inhibit fibronectinbinding to collagen, as well as to pharmaceutical compositionscontaining these peptides, and methods for inhibiting fibronectinbinding to collagen using these peptides.

BACKGROUND OF THE INVENTION

Thrombospondin is a multi-functional protein capable of interacting withnumerous macromolecules, e.g. fibronectin (Lahav, et al., 1984, Cell31:253-262; Lahav, et al. 1984, Eur. J. Biochem. 145:151-156), heparin(Lawler, et al. 1981, Thromb. Res. 22:267-279) and collagen (Lahav, etal. 1984, Cell 31:253-262, Mumby, et al., 1984 J. Cell. Biol.98:646-652). All of these molecules are constituents of theextracellular matrix, suggesting that thrombospondin forms complexeswith other matrix components following its deposition into the matrix.

Gelder and Brown reported that thrombospondin inhibits interactions offibronectin with gelatin (Gelder, F. B. and Brown, S. T. 1987, J. Lab.Clin. Med. 110:548-557). Platelet thrombospondin and an unidentified butbiologically similar plasma protein were shown to inhibit thegelatin-binding activity of fibronectin; however, the domains orsequences of thrombospondin responsible for the interaction remainedunknown. The interactive region has been implicated to be a differentsite on fibronectin, than the fibrin binding domain (Homandberg, G. A.and Kramer-Bjerke, J. 1987, Thromb. Res. 48:329-335) and at least twodistinct domains of thrombospondin have been shown to bind fibronectin(Dardik, R. and Lahaw, J. 1989, Eur. J. Biochem. 185:581-588).Fibronectin and heparin compete for binding to the 27 kDa fragment ofthrombospondin suggesting that these two proteins share a common orclosely oriented binding site within the N-terminal domain ofthrombospondin. Thrombospondin has also been shown to disrupt focalcontact adhesions of endothelial cells attached to a fibronectin matrix(Murphy-Ullrich, J. E., and Hook, M. 1989, J. Cell. Biol.109:1309-1319). The mechanism of this effect is unknown, but inhibitionof the activity of thrombospondin by sulfated polysaccharides suggestedthat the heparin-binding domain of thrombospondin is involved.

A number of biologically active peptides from thrombospondin have beenidentified and isolated. Two peptides, ArgGlyAspAla (Lawler, J. andHynes, R. O. 1986, J.Cell Biol. 103:1635-1641) and ValThrCysGly (Prater,et al., 1991, J.Cell Biol. 112: 1031-1040) are proposed to be ligandsfor the interaction of thrombospondin with protein receptors on the cellsurface. The thrombospondin peptide TrpSerProTrpSer (Guo, et al. 1992,Proc. Nat. Acad. Sci. USA, 89:3040-3044) binds to heparin and sulfatide.However, there are no known thrombospondin peptides that are capable ofbinding to fibronectin or related collagen binding proteins.

Fibronectin has been implicated in a variety of cell contact processes,including cell attachment and migration. Fibronectin interacts withcollagen through the "gelatin binding domain" of fibronectin and thisinteraction between collagen and fibronectin is fundamental to theorganization of extracellular matrices and the behavior of these cellson substrates (Vaberi, et al., 1978, Proc. Natl. Acad. Sci. USA, 75:4944-4948). Fibronectin is essential for the attachment and migration ofmany cells, including various tumor and cancer cells.

Accordingly, there is a need for inhibitors of fibronectin that willbind to fibronectin or related collagen-binding proteins with highaffinity. There is particularly a need for such inhibitors which willbind to fibronectin or related collagen-binding proteins to prevent thefibronectin-dependent cell adhesion to collagen matrices and to inhibitinteraction with collagens of other proteins that share homologies withthe gelatin-binding domain of fibronectin.

It is, therefore, an object of the present invention to provide highlyeffective peptides having sequences which bind specifically and withspecific affinity to fibronectin or other proteins having homologieswith the fibronectin gelatin-binding domain so as to inhibitfibronectin-dependent cell adhesion to collagen matrices and to inhibitinteraction of other proteins having homologies with the fibronectingelatin-binding domain with collagens.

It is a further object of the present invention to providepharmaceutical compositions containing at least one peptide which has ahigh affinity to fibronectin or other protein having homologies with thefibronectin gelatin-binding domain.

It is yet another object of the present invention to provide a methodfor binding fibronectin or other proteins having homologies with thefibronectin gelatin-binding domain in a patient in need thereof.

These and other objects of the invention will become more apparent fromthe following description and preferred embodiments and the aid of theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the binding of fibronectin to various immobilizedthrombospondin peptides, i.e. SerHisTrpSerProTrpSerSer (SEQ ID NO:8) (); LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerProTrpSerSer(SEQ ID NO:7) (∘);LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerPro (SEQ ID NO:5) ( );GlyGlyTrpSerHisTrp (SEQ ID NO:1) (Δ); and GlyGlyTrpSerHisTrpSerPro (SEQID NO:4) ( ).

FIG. 2 is a graph of the specificity of inhibition of fibronectinbinding to the thrombospondin peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1)by synthetic peptides. In FIG. 2(A) the peptides are GlyGlyTrpSerHisTrp(SEQ ID NO:1) ( ); GlyGlyTrpSerLysTrp (SEQ ID NO:9) ( );GlyGlyTyrSerHisTrp (SEQ ID NO:10) ( ); and GlyGlyTrpSerHisTyr (SEQ IDNO:11) ( ); FIG. 2(B): GlyGlyTrpSerHisTrp (SEQ ID NO:1) ( );GlyGlyTrpThrHisTrp (SEQ ID NO:12) ( ); GlyGlyTrpAlaHisTrp (SEQ ID NO:13)( ); GlyTrpSerHisTrp (SEQ ID NO:14) (+) and AspGlyTrpSerHisTrp (SEQ IDNO:15) (□).

FIG. 3 is a graph of the inhibition of fibronectin binding tothrombospondin peptide GlyGlyTrpSerHisTrp by soluble fibronectin,thrombospondin and peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1),GlyGlyTrpSerHisTrp (SEQ ID NO:1) ( ), fibronectin ( ) and thrombospondin( ).

FIG. 4 is a graph of the inhibition of fibronectin binding tothrombospondin peptide GlyGlyTrpSerHisTrp by proteolytic fragments offibronectin: 30 kDa gelatin-binding domain ( ), 31 kDa fibrin-bindingdomain (∘), 40 kDa heparin-binding domain ( ), 120 kDa cell-bindingdomain (Δ) and intact fibronectin ( ).

FIG. 5 is a graph of the inhibition of fibronectin binding to gelatin bypeptide GlyGlyTrpSerHisTrp (SEQ ID NO:1).

FIG. 6(A) is a bar graph of the inhibition of fibronectin-mediatedmelanoma cell A2058 adhesion to gelatin by peptides GlyGlyTrpSerHisTrp(SEQ ID NO:1) ( ); GlyGlyTrpSerLysTrp (SEQ ID NO:9) orTrpSerHisTrpSerPro (SEQ ID NO:2).

FIG. 6(B) is a bar graph of the inhibition of direct adhesion of A2058cells to immobilized fibronectin by the peptides of this invention.

FIG. 7(A) is a bar graph of the inhibition of fibronectin-mediatedcarcinoma cell MDA 4355 adhesion to gelatin by peptidesGlyGlyTrpSerHisTrp (SEQ ID NO:1); GlyGlyTrpSerLysTrp (SEQ ID NO:9) orTrpSerHisTrpSerPro (SEQ ID NO:2).

FIG. 7(B) is a bar graph of the inhibition of direct adhesion of MDA435s cells to fibronectin by the peptides.

FIG. 8 is a graph of the inhibition of fibronectin-mediated adhesion ofbreast carcinoma cells MDA 435s to native type 1 collagen by peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1); GlyGlyTrpSerLysTrp (SEQ ID NO:9);TrpSerHisTrpSerPro (SEQ ID NO:2) or GlyArgGlyAspSer (SEQ ID NO:16).

FIG. 9 is a bar graph of the inhibition of gelatinase activity bypeptide (SEQ ID NO:1).

SUMMARY OF THE INVENTION

The present inventors have isolated, purified and characterized orsynthesized and characterized biologically active peptides having aspecific binding affinity for the gelatin-binding domain of fibronectin,which peptides have a sequence of at least five amino acids includingthe sequence HisTrp and at least one other tryptophan residue. In apreferred embodiment of the invention the peptide includes the sequenceXaaHisTrp, wherein Xaa is an amino acid selected from serine, threonineand alanine. More preferably, the peptide has a sequence including thehexapeptide GlyGlyTrpXaaHisTrp, wherein Xaa is defined above. In aparticularly preferred embodiment of the invention, the peptide is apeptide having one of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ IDNO:8. Most preferred is the hexapeptide GlyGlyTrpSerHisTrp (SEQ IDNO:1).

In another aspect of the invention, there is provided a pharmaceuticalcomposition for binding fibronectin or other related collagen-bindingproteins which includes an effective amount of at least one peptide ofthe invention and a pharmaceutically acceptable excipient or carrier. Ina preferred embodiment of the invention, the pharmaceutical compositionincludes an effective amount of one of the peptides selected from SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8 and a pharmaceutically acceptable carrier orexcipient. Most preferably, the pharmaceutical composition of theinvention is the hexapeptide having sequence SEQ ID NO:1.

In another aspect of the invention, there is provided a method forbinding fibronectin or other collagen-binding proteins in a patient inneed of such treatment wherein an effective amount of peptide having aspecific binding affinity for the gelatin-binding domain of fibronectinand which includes at least 5 amino acids, including the sequence HisTrpand at least one other tryptophan residue, is administered to suchpatient. In a preferred embodiment of the invention method, the peptideis a peptide having a sequence selected from SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQID NO:8. Most preferred is a method wherein the peptide is thehexapeptide SEQ ID NO:1.

The term "collagen-binding proteins" is used herein to describe proteinshaving a domain capable of specifically binding to or interacting withcollagen.

The term "gelatin-binding domain of fibronectin" is used herein todescribe the amino acid sequence of mammalian fibronectin which is thesite of interaction of mammalian fibronectin with gelatin. The "gelatin"is used to define denatured collagen.

The term "specific binding affinity for fibronectin" is used herein tomean at least 1% by weight binding of fibronectin to peptides undersaturating peptide conditions. The symbol (+) indicates binding of 1 to5%, (++) indicates from more than 5 to 50% binding and (+++) indicatesmore than 50% binding to fibronectin.

The one letter codes for amino acids and the corresponding three lettercodes which are used herein are as follows:

W=Trp

K=Lys

Y=Tyr

P=Pro

D=Asp

R=Arg

F=Phe

Q=Gln

E=Glu

T=Thr

G=Gly

S=Ser

A=Ala

DETAILED DESCRIPTION OF THE INVENTION

The peptides according to the invention are derived from the extracellular matrix protein thrombospondin. In particular, the peptides arederived from one of the domains that mediate interaction ofthrombospondin with the matrix adhesive protein fibronectin. Thepeptides of the present invention are derived from the second type 1repeat of thrombospondin. Thrombospondin is a modular adhesiveglycoprotein that contains three domains that have been implicated inthe attachment of cells to thrombospondin. One region of thrombospondin,consisting of a chymotryptic 50 kDa peptide contains the three type 1repeats of a 57 residue segment homologous to two malarial proteins(Lawler J. and Hynes, R. O. 1986, J. Cell. Biol. 103: 1635-1648). Theamino acid sequence of the second type 1 repeat is incorporated hereinby reference thereto.

The peptides of this invention interact directly with thegelatin-binding domain of fibronectin. The gelatin-binding domain offibronectin is necessary for the interaction of fibronectin withgelatin. Specific and preferred sequences of the peptides of theinvention are set forth in Table 1 and may be produced by any of avariety of art known methods. Such methods of peptide synthesis include,for example, solid phase peptide synthesis, genetic engineeringtechniques including the cloning of DNA encoding the peptide in anexpression vector, and the direct isolation of the peptide fromthrombospondin.

Table 1 also includes other peptides (FN Binding-) which while havinghomology to the peptides according to the invention (FN Binding +, ++,+++) do not include the essential His-Trp (H-W) sequence.

                  TABLE 1                                                         ______________________________________                                        Fibronectin binding to immobilized peptides.                                  Peptide                                                                             Seq. Id No:                                                                             Sequence           FN Binding                                 ______________________________________                                        239    8        SHWSPWSS           +                                          246    7        KRFKQDGGWSHWSPWSS  ++                                         256    6        GGWSHWSPWSS        ++                                         259   17        GGWSHASPWSS        -                                          263    5        KRFKQDGGWSHWSP     ++                                         266   27        KRFKQDGGASHASP     -                                          264    4        GGWSHWSP           +                                          285    3        WSHWS              +                                          292    2        WSHWSP             +                                          300    1        GGWSHW             +++                                        322   28        QDGGWS             -                                          317   15        DGWSPW             -                                          318   21        GGWGPW             -                                          319   29        GTWSEW             -                                          320   30        GFWSEW             -                                          ______________________________________                                    

The peptides of the invention bind specifically to the gelatin-bindingdomain of fibronectin. The sequences responsible for fibronectin-bindingof the present peptides are contained in a peptide previously identifiedas a heparin-binding peptide, however, the optimal sequence for bindingfibronectin has only weak heparin-binding activity (U.S. applicationSer. No. 07/801,812). More particularly, a 17 amino acid peptide(peptide 246) (SEQ ID NO:7) comprising part of the second type I repeatof thrombospondin (KRFKQDGGWSHWSPWSS) exhibits strong affinity forfibronectin and is a potent inhibitor of fibronectin binding to gelatinand of fibronectin-mediated cell adhesion to a gelatin matrix. Bindingof the present peptides to fibronectin does not require the aminoterminal basic residues of peptide 246, since peptide 256 (GGWSHWSPWSS)(SEQ ID NO:6) which lacks these residues also binds fibronectin.

At least two tryptophan residues in the correct position are requiredfor binding. The central tryptophan residue of peptide 246 (SEQ IDNO:18) (residue 9) is required for binding since a similar peptide (259)lacking this tryptophan residue fails to bind (Table 1 and FIG. 1).Among overlapping peptides derived from the 17 amino acid peptide 246(SEQ ID NO:7) the peptide having the sequence GlyGlyTrpSerHisTrp (SEQ IDNO:1) has strongest binding activity (FIG. 1 and Table 1). Mostpreferred among the peptides of the invention is the hexapeptide havingthe sequence GlyGlyTrpSerHisTrp (SEQ ID NO:1).

The peptides of the present invention may contain sequences derived fromthe consecutively occurring human thrombospondin amino acids.Preferably, the consecutively occurring amino acid sequences areselected from amino acids 394-420 and 424-450. Especially preferred arethe consecutive amino acids 410-420 and 424-434.

The binding activity of several peptides derived from the second Type 1repeat of thrombospondin are shown in FIG. 1. At saturating peptideconcentrations of 100 μg/ml, more than 50% of added fibronectin is boundto the immobilized peptides of this invention. The hexapeptideconsisting of the peptide sequence GlyGlyTrpSerHisTpr (SEQ ID NO:1)binds fibronectin better than larger peptides containing the samesequence or intact thrombospondin, indicating that the 6 residuesequence is partially cryptic in thrombospondin. It is not necessarythat the peptides of the invention contain the entire hexapeptidesequence for effective fibronectin binding however, it is necessary thatthe peptides share some sequence homology with the hexapeptide. Twotryptophan residues and the His residue of the hexapeptide are essentialin the peptides of the invention since even conservative substitutionwith similar amino acids renders the peptides inactive (FIG. 2).Preferably, the serine residue is present but it is not essential.Optimal binding is obtained with peptides comprising the serine residue(FIG. 2B). Conservative substitution of the serine residue with athreonine residue decreases activity approximately five-fold andsubstitution of the serine residue with an alanine residue produces anactive peptide having a slightly higher inhibition constant than thethreonine analog. The presence of two glycine residues strongly enhancesactivity relative to a peptide analog having only one glycine residue(FIG. 2B). Conservative substitution of both glycine residues withalanine or of the first glycine residue with aspartic acid abolishesactivity. Preferably, the peptide comprises two glycine residues,however, peptides lacking one or both glycine residues are within thescope of the invention.

Accordingly, the peptides of this invention have a sequence of at least5 amino acids residues, which include at least two tryptophan residues,at least one of which is present as the sequence HisTrp (HW) whereinsaid peptide has specific binding affinity for the gelatin-binding siteof fibronectin. More preferred is a peptide with at least 5 amino acidresidues including the sequence XaaHisTrp, wherein Xaa is an amino acidselected from serine, threonine and alanine and wherein the 5 aminoacids has at least two Trp residues. Even more preferred is thehexapeptide GlyGlyTrpXaaHisTrp, wherein xaa is serine, threonine oralanine.

In a particularly preferred embodiment of the invention, the peptideaccording to the invention is a peptide having a sequence independentlyselected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. Most preferable is thepeptide SEQ ID NO:1.

The peptides of the present invention retain proper conformation insolution for binding to fibronectin with specific binding affinity.Moreover, the peptides of the present invention bind specifically to thegelatin-binding site of fibronectin and preferably, at least 5% bindingaffinity under saturating pending conditions. The present peptides arepotent inhibitors of fibronectin binding to gelatin and offibronectin-mediated cell adhesion to a gelatin matrix.

Fibronectin is an important extracellular matrix component that mediatescell interactions and extracellular matrix assembly during normaldevelopment. It also participates in homeostasis and wound healing, andpeptides that inhibit fibronectin interactions with cells have beendemonstrated to inhibit tumor metastasis in animal models (Humphries, M.J. Olden, K and Yamada, K., (1986) Science 233, 467-470).

The present peptides represent a new class of inhibitors of fibronectinfunction. The peptides of this invention bind specifically tofibronectin and inhibit interactions of fibronectin with collagen butnot with cell surface integrin receptors for fibronectin. As such, thesepeptides are applicable in regulating cell matrix interactions involvedin tumorigenesis, metastasis, wound repair and homeostasis. For example,the present peptides inhibit fibronectin-dependent adhesion of the humanbreast carcinoma cell line MDA-MB435 to immobilized gelatin. Theinhibition of adhesion of these cancer cells by the peptides of thepresent invention is specific in that a homologous peptide lackingfibronectin binding activity fails to inhibit adhesion (see FIGS. 6-A,6-B and 7).

As noted above, only partial sequence homology with the hexapeptideGlyGlyTrpSerHisTrp (SEQ ID NO:1) is needed to inhibit fibronectinbinding to gelatin. The existence of homologous sequences comprisingactive sites in various proteins suggests that the activities of otherproteins containing domains homologous to the fibronectingelatin-binding domain can be regulated with the peptides of the presentinvention. For example, such proteins as matrix metalloproteinase-2 andmatrix metalloproteinase share homology with the fibranectingelatin-binding domain and, therefore, may be regulated by the peptidesof this invention.

There is a high degree of conservation of the fibronectin-bindinghexapeptide GlyGlyTrpSerHisTrp (SEQ ID NO:1) in the thrombospondin genefamily. The sequence is completely conserved in the second type Irepeats of mouse and human Thbs1 genes and in Thbs 2 genes from mouseand chicken (Laherty, et al. 1992, J. Biol. Chem. 267: 3274-3281). Asearch of the Swiss-Prot data base using the search sequenceGlyGlyTrpSerHisTrp (SEQ ID NO:1) yielded several other proteins withsimilar sequences. (see Table II). Epstein-Barr virus ribonucleotidereductase contains the most homologous sequences, GlyGlyTrpPheHisTrp(SEQ ID NO:18). The α-subunit of the nicotinic acetylcholine receptor,which is highly conserved among vertebrate acetylcholine receptorsequences also shares sequence homology with the hexapeptide through thesequence GlyTrpLysHisTrp (SEQ ID NO: 19). A peptide of acetylcholinereceptor containing the sequence GlyTrpLysHisTrp (SEQ ID NO:19) plays arole in acetylcholine binding to the receptor. Furthermore anothersimilar sequence, TrpSerXTrpSer, has been identified as a potential"binding" sequence (Miyazaki, et al. 1991; Embo J. , 3191-3197;Yoshimura, et al. 1992, J. Biol. Chem. 267: 11619-11625). The ligandsthat interact with this site have not been identified, but loss ofreceptor processing or activity following mutation of the consensussequence suggests that cellular factors involved in protein folding,intracellular processing, or signal transduction are potential ligands.

                  TABLE II                                                        ______________________________________                                        Amino acid sequence homologies with the fibronectin-                          binding sequence from thrombospondin.                                         Source         Sequence                                                       ______________________________________                                        human Thbs1    KRFKQDGGWSHWSPWSSC                                             mouse Thbs1    KRFKQDGGWSHWSPWSSC                                             mouse Thbs2    TRIRQNGGWSHWSPWSSC                                             chicken Thbs2  HRIRQDGGWSHWSPWSSC                                             EBV ribonucleotide                                                                           KQSKYAGGWSHWHDWAGC                                             reductase                                                                     chicken AchR   WVMKDYRGWKHWVYYACC                                             T. California AchR                                                                           WVMKDYRGWKHWVYYTCC                                             rat AchR       WVIKEARGWKHWVFYSCC                                             mouse AchR     WVIKEARGWKHWVFYSCC                                             bovine AchR    WVIKESRGWKHWVFYACC                                             ______________________________________                                         Sequence identity with the thrombospondin sequence is indicated by bold       residues.                                                                

Compositions containing a therapeutically effective amount of at leastone peptide of the present invention are prepared by any methods knownin the art. For example, the compositions are prepared by forming anadmixture of at least one peptide of the present invention and apharmaceutically acceptable carrier or excipient, such as for example,sterile saline, Ringers solution, Ringer's lactate or 5% dextrose. Thepeptides may be mixed with a variety of carrier compounds depending onthe form of preparation desired for administration.

In another embodiment, the peptides of the present invention may beconjugated with a suitable carrier polymer or protein. For example,chemical conjugation to the peptide can be used to target specific cellsby attachment to an antibody against a surface protein of the targetcell.

Various methods of administration of peptides are known to those skilledin the art. Such methods of administration may include, but are notlimited to, surface application, oral and parenteral routes, injectioninto joints, subcutaneous injection via sustained release, intravenousinjection or other pharmaceutical methods of delivery.

Appropriate dosages of the peptides of the invention will depend uponthe condition presented by the individual subject. The skilled medicalworker will be able to determine appropriate dosages required to combatthe physiological activity. However, in general, amounts of from about 1μg to 100 μg/kg body weight/day of the biologically active peptideshould be useful.

The following examples are provided to more fully illustrate theprinciples and practices of the invention. The examples are not intendedin any way to limit the scope of the invention.

EXAMPLE 1

This example illustrates the binding of peptides of the invention tofibronectin.

Fibronectin was isolated from platelet-depleted plasma as described(Ariyama, et al. 1985, J. Biol. Chem. 260:4492-4500) and iodinated usingIodogen (Pierce Chemical Co., Rockford, Ill.) as described (Roberts, etal. 1987, J. Cell. Biol. 104:131-139).

Peptides were synthesized corresponding to sequences of humanthrombospondin deduced from a cDNA sequence for human endothelial cellthrombospondin (C Lawler and Hynes, 1986, J. Cell. Biol. 103:1635-1648).Peptides were synthesized on a Biosearch Model 9600 peptide synthesizerusing standard Merrifield solid phase synthesis protocols and t-Bocchemistry. Peptides were analyzed and where necessary, further purifiedbyreverse-phase HPLC chromatography. Identities of some of the peptides,including peptide 246 (SEQ ID NO:7), were verified by complete aminoacid sequence analysis. Peptide solutions were neutralized by additionof dilute NaOH and stored in solution at -20° C.

Direct binding of the fibronectin to immobilized peptides was carriedout by first absorbing the peptides on polyvinyl chloride microtiterwells for3 hours at 25° C. at the indicated concentrations. Unboundpeptides were removed by washing and wells were incubated for 30 minutesin tris-BSA (50 mMtris-HCl, pH7-8, 110mmNaCl, 1% BSA) (Sigma fatty acidand globulin-free, 0.1% NAN₃). The wells were washed and incubated with30 ml of 0.5 μg/ml ¹²⁵ I-fibronectin for 2 hours at 25° C. The wellswere washed five times with Dulbecco's phosphate buffered salineand cutfrom the plate and bound radioactivity was counted.

The peptides listed in Table 1 were synthesized as described above andabsorbed to microtiter wells. Of the larger peptides tested from thetype I repeats or the carboxyl terminal domain of thrombospondin, onlypeptide 246 (SEQ ID NO:7) bound labelled fibronectin. The peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1) exhibited the strongest bindingactivity.This peptide sequence is located in the second type I repeat ofthe human ThbsI gene. Synthetic peptides 317 (SEQ ID NO:15) and 318 (SEQID NO:20), derived from the corresponding positions in the first andthird type I repeats of thrombospondin were inactive.

The results are shown in Table 1 and FIG. 1. FIG. 1 shows binding ofI-fibronectin to peptides 300 (Δ) (GlyGlyTrpSerhisTrp) (SEQ ID NO:1);246 (∘) (LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerProTrpSerSer) (SEQ IDNO:7); 263 () (LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerPro) (SEQ ID NO:5)() and 264 (GlyGlyTrpSerHisTrpSerPro) (SEQ ID NO:4 ).

EXAMPLE 2

This example illustrates the specificity of inhibition of fibronectinbinding to thrombospondin peptide 300 (GlyGlyTrpSerHisTrp) by syntheticpeptides.

Microtiter plate wells were coated with 50 μg/ml of peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1) as in Example 1. Binding of ¹²⁵I-fibronectin (0.5 μg/ml) to bound peptide was determined in thepresence of increasing amounts of the peptides: Panel A: the peptidesare GlyGlyTrpSerHisTrp (SEQ ID NO:1) (); GlyGlyTrpSerLysTrp (SEQ IDNO:9) (); GlyGlyTyrSerHisTrp (SEQ ID NO:10) () and GlyGlyTrpSerHisTyr(SEQ ID NO:11)(); Panel B: GlyGlyTrpSerHisTrp (SEQ ID NO:1) ();GlyGlyTrpThrHisTrp (SEQ ID NO:12) (); GlyGlyTrpAlaHisTrp (SEQ ID NO:13)(); GlyTrpSerHisTrp (SEQ ID NO:14) (+); AlaAlaTrpSerHisTrp (SEQ IDNO:21) (); and AspGlyTrpSerHisTrp (SEQ ID NO:22) (□) .

The results are shown in FIG. 2.

Binding of fibronectin to immobilized peptide GlyGlyTrpSerHisTrp (SEQ IDNO:1) was inhibited by soluble peptide 300 with 50% inhibition at 27 μm.Three hexapeptides containing conservative single amino acidsubstitutions, GlyGlyTrpSerLysTrp (SEQ ID NO:9); GlyGlyTyrSerHisTrp (SEQID NO:10) and GlyGlyTrpSerHisTyr (SEQ ID NO:11) were inactive. Thecorresponding hexapeptides from the first and third repeats ofthrombospondin were also inactive.

EXAMPLE 3

This example illustrates the inhibition of fibronectin binding tothrombospondin peptide GlyGlyTrpSerHisTrp by soluble fibronectin,thrombospondin and synthetic peptide GlyGlyTrpSerHisTrp.

Human platelet thrombospondin was purified according to the method ofRoberts, et al. 1985 J. Cell Biol. 104:131-139.

Binding of ¹²⁵ I-fibronectin (0.5 μg/ml) to microtiter plate wellscoated with 50 μg/ml of peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1) wasdetermined in the presence of synthetic peptide GlyGlyTrpSerHisTrp (SEQIDNO:1), fibronectin or thrombospondin. The results are shown in FIG. 3.

Unlabelled fibronectin and thrombospondin competed for binding of ¹²⁵I-fibronectin to the peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1), butseveralcontrol proteins, i.e. ovalbumin, transferrin, fetuin, goat IgGand murine laminin did not (IC₅₀ >200 μg/ml).

EXAMPLE 4

This example illustrates that peptides of the present invention andwhole thrombospondin bind to the gelatin-binding domain of fibronectin.

Microtiter plates were coated with 50 μg/ml of synthetic peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1) as described in Example 1. Binding of¹²⁵ I-fibronectin (0.5 μg/ml) to the bound peptide was determined in thepresence of proteolytic fragments of fibronectin, which were obtainedfrom Telios Pharmaceuticals, Inc. San Diego, Calif. The proteolyticfragments included intact fibronectin; 30 kDa gelatin-binding domain; 33kDa recombinant cell binding domain; 28 kDa recombinant cell bindingdomain and transferrin. The results are shown in FIG. 4.

Of the proteolytic fragments tested, the gelatin-binding domain was thestrongest inhibitor of ¹²⁵ I-fibronectin binding to immobilized peptide.The 33 kDa fragment, which contains the cell binding domain wasinhibitory. However, the 28 kDa fragment which contains all but theN-terminal 5 kDa of the 33 kDa fragment was inactive and a 40 kDafragmentwith the same amino terminus as the 28 kDa fragment wasapproximately 10-fold less active. Since the sequence of the 33 kDarecombinant fragmentis contained in the 120 kDa fragment of fibronectinand the 120 kDa fragment was only a weak inhibitor, the activityexpressed by the 33 kDa fragment is probably due to a cryptic site. Theactivity of the 33 kDa fragment is less than that of the gelatin bindingfragment.

EXAMPLE 5

This example illustrates the .specificity of inhibition of fibronectinbinding to gelatin by the peptide GlyGlyTrpSerHisTrp.

Wells of a microtiter plate were incubated for 3 hours at 25° C. with 2μg/ml gelatin in Dulbecco's PBS. The wells were emptied and incubatedwith Dulbecco's PBS containing 1% BSA for 30 minutes. The wells wereemptied and incubated with 0.5 μg/ml ¹²⁵ I-fibronectin in thepresence ofpeptide GlyGlyTrpSerHisTrp (SEQ ID NO:1) (); GlyGlyTrpSerLysTrp(SEQ IDNO:9) (); GlyGlyTyrSerHisTrp (SEQ ID NO:10) (); or GlyGlyTrpSerHisTyr(SEQ ID NO:11) () for 2 hours at 25° C. The wells were washed 4 timeswith PBS, cut from the plate and the bound radioactivity was counted.The results are shown in FIG. 5.

The inhibition by peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1) was specificin that the related peptides GlyGlyTrpSerLysTrp (SEQ ID NO:9);GlyGlyTyrSerHisTrp (SEQ ID NO:10) and GlyGlyTrpSerHisTyr (SEQ ID NO:11),with single amino acid substitutions from the active sequence wereinactive.

EXAMPLE 6

A ligand binding assay as described in Example 5 was performed exceptthat microtiter plates were coated with immobilized native type Icollagen in place of gelatin.

The peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1) inhibited fibronectinbinding to type I collagen with an IC₅₀ =40 μm. GlyGlyTrpAlaHisTrp (SEQIDNO:13) was a weak inhibitor and the peptides TrpSerHisTrpSerPro (SEQID NO:2); GlyGlyTrpSerLysTrp (SEQ ID NO:9); GlyGlyTyrSerHisTrp (SEQ IDNO:10); GlyGlyTrpSerHisTyr (SEQ ID NO:11); AlaAlaTrpSerHisTrp (SEQ IDNO:21); AspGlyTrpSerHisTrp (SEQ ID NO:22); and GlyGlyTrpThrHisTrp (SEQID NO:24) were inactive.

EXAMPLE 7

This example illustrates the inhibition of fibronectin-dependent celladhesion to denatured collagen by peptide GlyGlyTrpSerHisTrp (SEQ IDNO:1).

Gelatin (1 μg/ml) in Dulbecco's PBS was coated on plastic discs in 24well plates and incubated for 2 hours at 37° C. After the supernatantwas removed, the discs were treated with 1% BSA-tris pH 7.8 for 30minutes at ambient temperature. The discs were washed twice with PBS and0.4 ml of RPM1 1640 medium containing 0.1% BSA and 1 μg/ml fibronectinand 2, 20 or 200 μg of the inhibitory peptides were added to the wells.A suspension of 10⁵ cells in 0.1 ml RPMI medium containing 0.1% BSA wasadded to the wells and incubated at 37° C. with 5% CO₂ for 1 hour. Thediscs were washed to remove nonadherent cells and stained. Adherentcells were counted microscopically. The results are shown in FIGS. 6 and7.

In panel A of FIG. 6 human melanoma cell of cell line A2058 were added.to the wells and in Panel A of FIG. 7 MDA 435s breast carcinoma cellswere added to immobilized gelatin. At the concentrations used, the cellsweaklyadhered to gelatin in the absence of added fibronectin. Thestimulated adhesion in the presence of fibronectin was inhibited in adose-dependent manner by the peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1).The inhibition wasspecific in that the homologous peptides that lackedfibronectin binding activity, GlyGlyTrpSerLysTrp (SEQ ID NO:9) andTrpSerHisTrpSerPro (SEQ ID NO:2), were inactive.

Direct adhesion of A2058 cells (FIG. 6B) or MDA 435s cells (FIG. 7B) toimmobilized fibronectin was not inhibited by the peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1) or the control peptides, but adhesionof the breast carcinoma cells to fibronectin was inhibited by thepeptide GlyArgGlyAspSer (SEQ ID NO:25). Thus, interaction of fibronectinwith integrin receptors on the breast carcinoma cells is not inhibitedby the peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1).

EXAMPLE 8

This example illustrates the inhibition of fibronectin-mediated adhesionofbreast carcinoma cells to native type I collagen by peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1).

Discs were treated as in Example 7 except that 1 μg/ml of type Icollagen in Dulbecco's PBS was coated on the discs. Cell attachment ofbreast carcinoma cells (cell line MDA 435s) to immobilized type Icollagenwas determined in the presence of fibronectin alone and in thepresence of peptide GlyGlyTrpSerHisTrp (SEQ ID NO:1); GlyGlyTrpSerLysTrp(SEQ ID NO:9); TrpSerHisTrpSerPro (SEQ ID NO:2) or GlyArgGlyAspSer (SEQID NO:25).The results are shown in FIG. 8.

Fibronectin-mediated adhesion of breast carcinoma cells to native type 1collagen was inhibited by peptides GlyGlyTrpSerHIsTrp (SEQ ID NO:1) andGlyArgGlyAspSer (SEQ ID NO:25) but not GlyGlyTrpSerLysTrp (SEQ ID NO:9)orTrpSerHisTrpSerPro (SEQ ID NO:2). Direct adhesion to type 1 collagenwas stronger than to gelatin. The effects of the peptideGlyGlyTrpSerHisTrp (SEQ ID NO:1) were specific forfibronectin-stimulated adhesion, however, this peptide does not inhibitdirect binding of cells to type 1 collagen.

EXAMPLE 9

This example illustrates inhibition of gelatinase by peptideGlyGlyTrpSerHisTrp. Degradation of ¹²⁵ I-gelatin by gelatinase activityin conditioned medium from bovine corneal endothelial cells wasdetermined by release of radioactivity from microtiter plate wellscoated with the radiolabeled substrate and incubated for 60 or 90 minwith conditioned medium activated using p-hydroxymercuribenzoate.Activity in the presence of the indicated concentrations of peptideGlyGlyTrpSerHisTrp(SEQ ID NO:1) is presented as a percent of controlactivity determined in the absence of peptide and corrected forbackground release determined in the absence of mercurial activation.Inhibition was also determined by a gelatin zymogram of endothelial cellconditioned medium: a, control; b, 50 μg/ml GlyGlyTrpSerHisTrp (SEQ IDNO:1); c, 500 μg/ml GlyGlyTrpSerHisTrp (SEQ ID NO:1); d, 50 μg/mlGlyGlyTrpSerLysTrp (SEQ ID NO:9); e, 500 μg/ml GlyGlyTrpSerLysTrp; andf, 500 μg/ml peptideLysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerProTrpSerSer(SEQ ID NO:7).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES:30                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acid                                                           (C) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:Peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1:                                        GlyG lyTrpSerHisTrp                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (C) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 2:                                       TrpSerHisTrp SerPro                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:5 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 3:                                       TrpSerHisTrpSer                                                                15                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:8 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 4:                                       GlyGlyTrpSerHisTrpSerPro                                                       15                                                                           (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:14 amino acids                                                     (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 5:                                       LysArgPheLysGlnAspGlyGlyTrpSerH isTrpSerPro                                   1510                                                                          (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:11 amino acids                                                     (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 6:                                        GlyGlyTrpSerHisTrpSerProTrpSerSer                                            1510                                                                          (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:17 amino acids                                                     (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 7:                                       LysArgPheLysGlnAspGlyGlyTrpSerHisTrpSerPro                                    1510                                                                          TrpSerSer                                                                     15                                                                             (2) INFORMATION FOR SEQ ID NO: 8:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:8 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 8:                                       SerHisTrpSerProTrpSerSer                                                      15                                                                            (2 ) INFORMATION FOR SEQ ID NO: 9:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 9:                                       GlyGlyTrpSerLysTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 10:                                      GlyGlyTyrSerHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 11:                                      GlyGlyTrpSerHisTyr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH:6 amino acids                                                     (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 12:                                      GlyGlyTrpThrHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 13:                                      GlyGlyTrpAlaHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                      (B) TYPE:amino acids                                                         (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 14:                                      GlyTrpSerHisTrp                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                           (D) TOPOLOGY:linear                                                          (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 15:                                      AspGlyTrpSerHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:5 amino acids                                                      (B) TYPE:amino acids                                                          (D ) TOPOLOGY:linear                                                          (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 16:                                      GlyArgGlyAspSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:10 amino acids                                                     (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 17:                                      GlyTrpSerHisAlaSerProTrpSerSer                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                           (D) TOPOLOGY:linear                                                          (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 18:                                      GlyGlyTrpPheHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE:amino acids                                                           (D) TOPOLOGY:linear                                                          (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 19:                                      GlyTrpLysHisTrp                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                            (ii) MOLECULE TYPE:peptide                                                   (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 20:                                      GlyGlyTrpGlyProTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                     (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 21:                                     AlaAlaTrpSerHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 22:                                      AspGlyTrpSerHisTrp                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 23:                                       TrpSerHisTrpSerPro                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 24:                                       GlyGlyTrpThrHisTrp                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:5 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 25:                                      GlyArg GlyAlaSer                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:14 amino acids                                                     (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 26:                                      LysArgPheLysGlnAs pGlyGlyAlaSerHisAlaSerPhe                                   1510                                                                          (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 27:                                      GlnAspGlyGlyTrpSer                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 28:                                       GlyThrTrpSerGluTrp                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:6 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 29:                                       GlyPheTrpSerGluTrp                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:7 amino acids                                                      (B) TYPE:amino acids                                                          (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 30:                                      GlyGlyT rpSerLysSerTrp                                                        15                                                                        

What is claimed is:
 1. A method for binding fibronectin comprisingcontacting fibronectin with an effective amount of a compositioncomprising a peptide having a specific binding affinity for thegelatin-binding domain of fibronectin, said peptide having from 3 to 18amino acids in length and consisting of the sequence (A),

    Xa--Xb--His--Trp--Xc,                                      (A)

wherein Xa is H or an amino acid sequence of from 1 to 13 amino acidsderived from the consecutively occurring thrombospondin amino acidresidues 394-420, Xb is serine, alanins, tryptophan, or threonine and Xcis OH, NH₂, or an amino acid sequence of from I to 13 amino acidsderived from the consecutively occurring human thrombospondin amino acidresidues 424-450, and at least one of Xa and Xb includes a tryptophanresidue located within three amino acid residues from Trp in sequence(A).
 2. A method according to claim 1 wherein said method is conductedin vitro or in vivo.
 3. A method according to claim 1 wherein saidmethod is conducted in vitro.
 4. A method according to claim 1 whereinsaid method is conducted in vivo.
 5. A method according to claim 4wherein said method is conducted in a patient.
 6. A method for bindingfibronectin, according to claim 5, wherein said peptide is a peptidehaving a sequence selected from the group consisting of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8.
 7. A method for inhibiting enzymatic activity ofcollagenase in a patient in need of such treatment, which comprisesadministering to said patient an effective amount of at least onepeptide contained in a pharmaceutically acceptable excipient or carrier,wherein said peptide is a peptide having a sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
 8. A methodaccording to claim 5 wherein Xb is selected from the group consisting ofserine, alanine and tryptophan.
 9. The method according to claim 7wherein said collagenase is endothelial collagenase.
 10. A methodaccording to claim 6 wherein said peptide is a peptide having SEQ IDNo:1.
 11. A peptide composition having a specific binding affinity forfibronectin, consisting essentially of a peptide selected from the groupconsisting of peptides having SEQ ID NO:1, SEQ ID NO:12 and SEQ IDNO:13.
 12. A method according to claim 10, wherein said peptide furthercomprises an amino-terminal N-acetyl and carboxyl terminal amide.